The Roles Of CCR8 Positive Treg Cell Infiltration In Promoting Immune Evasion In Muscle-invasive Bladder Cancer

Objective:Intratumoral regulatory T cells(Tregs)represent a major barrier to effective anti-tumor immunity.Identifying pathways that could be targeted to selectively undermine intratumoral Tregs is essential.CCR8 has been identified as an important chemokine receptor on intratumoral Tregs and is known to be critical for CCR8~+Treg-mediated immunosuppression.This study aimed to investigate the clinical significance and inherent molecular mechanisms of intratumoral CCR8~+Tregs in Muscle-invasive bladder cancer(MIBC).Methods:The accumulation of CCR8~+Tregs in MIBC was identified via immunohistochemistry(IHC)and immunofluorescence(IF).259 MIBC tissues from two independent clinic centers were included and analyzed for the prognostic merit of CCR8~+Tregs via IHC.83 fresh MIBC tumor tissues were used to evaluate the proportion and function of immune cells in MIBC via flow cytometry and ex vivo intervention experiments.396 MIBC patients of the Cancer Genome Atlas(TCGA)were applied for bioinformatics analysis.Results:It was found that MIBC samples displayed a higher percentage of Tregs expressing CCR8 compared with peritumor tissues.The number of CCR8~+Tregs was apparently correlated with enhanced tumor stages based on the IHC staining.Patients with high CCR8~+Tregs had significantly poorer overall survival(OS)and recurrence-free survival(RFS).It was found that CCR8~+Treg was an independent adverse prognosticator for OS and RFS according to multivariate analysis in all patients.The interaction test between CCR8~+Tregs and ACT responsiveness revealed that CCR8~+Tregs low patients exhibited far better therapeutic responsiveness to ACT than CCR8~+Tregs high patients.It was shown that the CCR8expression by intratumoral Tregs maintained the stability and potentiated their suppressive function by upregulating the expression of transcript factors FOXO1 and c-MAF.Through flow cytometry analysis,CD8~+T cells in MIBC specimens with high CCR8~+Tregs displayed exhausted phenotype with decreased IFN?,TNF?and Ki67,yet elevated PD-1 and TIGIT expression.While CD4~+T cells in MIBC specimens with high CCR8~+Tregs exhibited less proinflammatory phenotype with decreased IFN?and IL17A.Moreover,it was revealed that CCR8 blockade using neutralizing antibody can destabilize intratumoral Tregs into a fragile phenotype,reactivate the antitumor immunity and augment the therapeutic benefits of the anti-PD-1antibody in MIBC.Conclusions:This study identified CCR8~+Tregs as a stable Treg subtype and an independent prognostic indicator for survival and responsiveness to ACT in patients with MIBC.Intratumoral CCR8~+Tregs infiltration defined immunoevasive contexture with enhanced immune tolerance of CD8~+T cells and weakened proinflammatory phenotype of CD4~+T cells.Most importantly,therapeutic targeting of CCR8 provided a more specific modulation of Tregs and a potentially robust approach to MIBC immunotherapy.